Method for production of polypeptide

ABSTRACT

The present invention provides a method capable of producing a natural or recombinant protein in high yield. 
     The present invention relates to a method of producing a polypeptide, comprising culturing a cell which strongly expresses cysteine sulfinic acid decarboxylase and has a transferred DNA encoding a desired polypeptide and thereby allowing the cell to produce the polypeptide. Hamster cysteine sulfinic acid decarboxylase, a DNA encoding the same, a recombinant vector and a transformed cell are also provided.

TECHNICAL FIELD

The present invention relates to a method of producing a polypeptide, more specifically, a method of producing a polypeptide using a cell which strongly expresses cysteine sulfinic acid decarboxylase.

BACKGROUND ART

When proteins useful as pharmaceuticals are produced with the recombinant DNA technique, use of animal cells enables complicated post-translational modification and folding which prokaryotic cells can not perform. Therefore, animal cells are frequently used as host cells for producing recombinant proteins.

Recently, a large number of biopharmaceuticals, such as antibodies and physiologically active proteins, have been developed. Techniques that permit efficient production of recombinant proteins by animal cells lead to cost reduction of biopharmaceuticals and promise their stable supply to patients.

Under these circumstances, a method of protein production with higher production efficiency is desired.

β-alanine, by itself, has pH-buffering action and antioxidant action. Such β-alanine is also a precursor of stronger carnosine (Non-Patent Document 1).

In addition, it has been known that β-alanine functions as a signal peptide, organic osmolyte (Non-Patent Documents 2 and 3), and that it plays a role in maintaining enzyme activity by its chaperone-like activity (Non-Patent Document 4).

On the other hand, it has never been known that an increase in the concentration of β-alanine in cells contributes to the improvement of production of a desired recombinant protein in cultured cells.

As a β-alanine synthetase found in the brain, glutamate decarboxylase (GAD) (EC 4.1.1.15) (Non-Patent Document 5) has been known. GAD is also a decarboxylase having activity for synthesizing hypotaurine or taurine. In general, enzymes known to be used in the synthesis of taurine from a sulfur-containing amino acid such as cysteine (Non-Patent Documents 6 and 7) include cysteine sulfinic acid decarboxylase (CSAD) (EC 4.1.1.29) and cysteine dioxygenase (CDO) (EC 1.13.11.20). However, it has not been known that a large amount of β-alanine is produced if cysteine sulfinic acid decarboxylase is strongly expressed in CHO cells. Further, hamster cysteine sulfinic acid decarboxylase and hamster cysteine dioxygenase have not been known, either.

[Non-Patent Document 1]

-   Babizhayev, M. A., Biochim. Biophys. Acta (1989) 1004, 363-371

[Non-Patent Document 2]

-   Hammer, M. A., Baltz, J. M., Mol. Reprod. Dev. (2003) 66(2), 153-61

[Non-Patent Document 3]

-   Mariapia, V, et. al. Biochim. Biophys. Acta (2002) 1587, 83-91

[Non-Patent Document 4]

-   Mehta, A. D., Seidler, N. W., Journal of Enzyme Inhibition and     Medicinal Chemistry (2005) 20(2), 199-203

[Non-Patent Document 5]

-   Kelly, C., Carter, N. D., Johnstone, A. P. and Nussey, S. S.,     Lancet (1991) 338 (8780), 1468-1469

[Non-Patent Document 6]

-   M. L. Tappaz, Neurochemical Research, Vol. 29, No. 1, January 2004,     pp. 83-96

[Non-Patent Document 7]

-   Ian Henry Lambert, Neurochemical Research, Vol. 29, No. 1, January     2004, pp. 27-63

DISCLOSURE OF THE INVENTION Problem for Solution by the Invention

It is an object of the present invention to provide a method which is capable of producing a natural or recombinant protein in high yield.

Means to Solve the Problem

As a result of extensive and intensive researches toward the solution of the above problem, the present inventors have found that it is possible to increase the yield of a desired polypeptide by using a cell which strongly expresses cysteine sulfinic acid decarboxylase. Thus, the present invention has been achieved. Moreover, the amount of a desired polypeptide produced could be further increased by using cells which strongly coexpresses cysteine sulfinic acid decarboxylase and a taurine transporter. Furthermore, the present inventors have found that such cells that strongly express cysteine sulfinic acid decarboxylase significantly increase the amount of β-alanine produced. Since it was anticipated that β-alanine would neutralize the fatigue substance lactate by its antioxidant action and bind it to redundant sugar in cells to eliminate them from body, an increase in the β-alanine concentration in the cells was considered to contribute to an increase in the amount of a desired polypeptide produced. Further, as a result of studies conducted by the present inventors, it was found that even when β-alanine was added to a medium for cell culture, the intracellular concentration of β-alanine could not be said to have increased, and thus that mere addition of β-alanine to a medium was insufficient.

The present invention may be summarized as follows.

(1) A method for producing a polypeptide, in which cells capable of high-yield production of β-alanine and into which DNA encoding a desired polypeptide has been transferred are cultured to produce the desired polypeptide. (2) The production method of (1) above, wherein the cells capable of high-yield production of β-alanine are cells into which DNA encoding a protein having enzyme activity for synthesizing β-alanine from aspartic acid has been transferred. (3) The production method of (1) above, wherein the cells capable of high-yield production of β-alanine are cells into which DNA encoding cysteine sulfinic acid decarboxylase, glutamate decarboxylase, or a mutant thereof has been transferred. (4) The production method of any one of (1) to (3) above, wherein the cells capable of high-yield production of β-alanine are cells in which the ratio of the intracellular concentration of β-alanine on the 6^(th) or subsequent days after the initiation of the culture to that at the initiation of the culture is 2 or greater. (5) The production method of any one of (1) to (3) above, wherein the cells capable of high-yield production of β-alanine are capable of increasing the β-alanine concentration in the culture medium on 6^(th) or subsequent days after the initiation of the culture by 50 μM or more. (6) A cultured cell, in which the ratio of the intracellular concentration of β-alanine on the 6^(th) or subsequent days after the initiation of the culture to that at the initiation of the culture is 2 or greater. (7) A cultured cell, which is capable of increasing the β-alanine concentration in the culture medium on the 6^(th) or subsequent days after the initiation of the culture by 50 μM or more. (8) A method for producing a polypeptide, in which cells that strongly express cysteine sulfinic acid decarboxylase and into which DNA encoding a desired polypeptide has been transferred are cultured to produce the desired polypeptide. (9) The production method of (8) above, wherein the cells that strongly express cysteine sulfinic acid decarboxylase are cells into which DNA encoding cysteine sulfinic acid decarboxylase has been transferred. (10) The production method of (8) or (9) above, wherein the cells that strongly express cysteine sulfinic acid decarboxylase further express a taurine transporter strongly. (11) The production method of (10) above, wherein the cells that strongly express a taurine transporter are cells into which DNA encoding a taurine transporter has been transferred. (12) The method of (9) or (11) above, wherein the cell is Chinese hamster ovary cells. (13) The method of any one of (8) to (12) above, wherein the desired polypeptide is an antibody. (14) The method of any one of (9) to (13) above, wherein the DNA encoding the cysteine sulfinic acid decarboxylase is any one of the following (a) to (e):

(a) a DNA encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO: 2, 4, 6 or 8;

(b) a DNA encoding a polypeptide which has an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 2, 4, 6 or 8 by substitution, deletion, addition and/or insertion of one or more amino acid residues and yet has cysteine sulfinic acid decarboxylase activity;

(c) a DNA encoding a polypeptide having 70% or more amino acid sequence homology with the amino acid sequence as shown in SEQ ID NO: 2, 4, 6 or 8 and yet having cysteine sulfinic acid decarboxylase activity;

(d) a DNA having the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5 or 7;

(e) a DNA which hybridizes to a DNA complementary to a DNA having the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5 or 7 under stringent conditions and yet encodes a polypeptide having cysteine sulfinic acid decarboxylase activity.

(15) A DNA encoding cysteine sulfinic acid decarboxylase, which is any one of the following (a) to (e), provided that DNA having the nucleotide sequence as shown in SEQ ID NO: 3, DNA having the nucleotide sequence as shown in SEQ ID NO: 5, and DNA having the nucleotide sequence as shown in SEQ ID NO: 7 are excluded:

(a) a DNA encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO: 2;

(b) a DNA encoding a polypeptide which has an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 2 by substitution, deletion, addition and/or insertion of one or more amino acid residues and yet has cysteine sulfinic acid decarboxylase activity;

(c) a DNA encoding a polypeptide having 97% or more amino acid sequence homology with the amino acid sequence as shown in SEQ ID NO: 2 and yet having cysteine sulfinic acid decarboxylase activity;

(d) a DNA having the nucleotide sequence as shown in SEQ ID NO: 1;

(e) a DNA which hybridizes to a DNA complementary to a DNA having the nucleotide sequence as shown in SEQ ID NO: 1 under stringent conditions and yet encodes a polypeptide having cysteine sulfinic acid decarboxylase activity.

(16) A polypeptide which is any one of the following (A) to (E), provided that a polypeptide having the amino acid sequence as shown in SEQ ID NO: 4, a polypeptide having the amino acid sequence as shown in SEQ ID NO: 6, and a polypeptide having the amino acid sequence as shown in SEQ ID NO: 8 are excluded:

(A) a polypeptide having the amino acid sequence as shown in SEQ ID NO: 2;

(B) a polypeptide which has an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 2 by substitution, deletion, addition and/or insertion of one or more amino acid residues and yet has cysteine sulfinic acid decarboxylase activity;

(C) a polypeptide having 97% or more amino acid sequence homology with the amino acid sequence as shown in SEQ ID NO: 2 and yet having cysteine sulfinic acid decarboxylase activity;

(D) a polypeptide encoded by a DNA having the nucleotide sequence as shown in SEQ ID NO: 1;

(E) a polypeptide encoded by a DNA which hybridizes to a DNA complementary to a DNA having the nucleotide sequence as shown in SEQ ID NO: 1 under stringent conditions and yet encodes a polypeptide having cysteine sulfinic acid decarboxylase activity.

(17) A recombinant vector comprising the DNA of (15) above. (18) A cell into which the DNA of (15) above has been transferred. (19) A cell which has a transferred DNA encoding cysteine sulfinic acid decarboxylase and a transferred DNA encoding a desired polypeptide. (20) The cell according to (19) above, which further has a transferred DNA encoding a taurine transporter. (21) A cell which has a transferred DNA encoding cysteine sulfinic acid decarboxylase and a transferred DNA encoding a taurine transporter. (22) A cell capable of high-yield production of β-alanine which can be obtained by transferring a DNA encoding cysteine sulfinic acid decarboxylase. (23) The cell of (22) above, which further has a transferred DNA encoding a desired polypeptide.

EFFECT OF THE INVENTION

According to the present invention, it has become possible to increase the yield of a desired polypeptide.

The present specification encompasses the contents disclosed in the specification and/or the drawings of Japanese Patent Application No. 2007-66172 based on which the present patent application claims priority.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence of a newly cloned, CHO cell-derived hamster CSAD gene and the amino acid sequence deduced therefrom.

FIG. 2 shows the nucleotide sequence of a newly cloned, CHO cell-derived hamster CDO1 gene and the amino acid sequence deduced therefrom.

FIG. 3 shows a plasmid which was used for expressing hamster CSAD (493 amino acids).

FIG. 4 shows a plasmid which was used for expressing hamster CDO1 (200 amino acids).

FIG. 5 shows anti-glypican-3 antibody yield plots on day 14 of 50 ml shaker flask fed-batch culture.

FIG. 6 shows a plasmid which was used for expressing Hamster CSAD (493 amino acids) in a TauT strongly expressing strain.

FIG. 7 is a graph showing the amounts of antibodies produced in the feeding cultures of four pPur/CSAD-transferred TauT strongly expressing strains and TauT strongly expressing cells as a parent strain in 50-ml shakers.

FIG. 8 is a graph showing the amount of lactate produced in the feeding cultures in 50-ml shakers. Suppressed the production of lactate was observed in the pPur/CSAD-transferred TauT strongly expressing strains.

FIG. 9 is a graph showing the survival ratio of the cells in the feeding cultures in 50-ml shakers. The survival ratio was maintained in the pPur/CSAD-transferred TauT strongly expressing strains.

FIG. 10 is a graph showing the amount of an antibody produced in the feeding culture in a 1-L jar of the pPur/CSAD-transferred TauT strongly expressing strain that shows the highest survival ratio in FIG. 9.

FIG. 11 is a graph showing the contents (concentration ratios) of intracellular alanine and intracellular β-alanine during feeding cultures in 1-L jars.

FIG. 11-1 is a graph showing the concentrations of alanine and β-alanine in the culture media during feeding cultures in 1-L jars.

FIG. 12 is a graph showing the survival ratio of the cells in feeding cultures in 1-L jars. The pPur/CSAD-transferred cells maintained their survival ratio in comparison with the parent strain.

FIG. 13 is a graph showing the content (concentration ratio) of intracellular ammonia during feeding cultures in 1-L jars. Ammonia generation in the pPur/CSAD-transferred TauT strongly expressing strain was significantly suppressed, as compared with the parent strain.

FIG. 14 is a graph showing the survival ratio of the cells in batch cultures in 50-ml shakers. As a result of addition of β-alanine (25 mM) to the parent strain, the survival ratio of the parent strain was maintained at levels equivalent to that of a CSAD-transferred strain capable of high-yield production of β-alanine.

FIG. 15 is a graph showing the survival ratio of the cells on the 6^(th) day of batch cultures in 50-ml shakers. As a result of addition of β-alanine (25 mM) to the TauT-transferred strain, the survival ratio of the TauT-transferred strain was maintained at a level equivalent to that of a TauT+CSAD-transferred strain capable of high-yield production of β-alanine.

FIG. 16 shows the nucleotide sequence of a cloned, CHO cell-derived hamster taurine transporter gene and the amino acid sequence deduced therefrom.

FIG. 17 is a taurine transporter membrane topology of a newly cloned, CHO cell-derived hamster TauT.

FIG. 18 shows a plasmid which was used for expressing hamster TauT (622 amino acids).

FIG. 19 shows viable cell density plots on day 7 of 50 ml shaker flask batch culture. The viable cell density in pHyg/TauT-transferred cell was superior to that in pHyg-transferred cell.

FIG. 20 shows lactate yield plots on day 7 of 50 ml shaker flask batch culture. pHyg/TauT-transferred cell produced less lactate, and was superior to pHyg-transferred cell.

FIG. 21 shows anti-glypican-3 antibody yield plots on day 7 of 50 ml shaker flask batch culture. Four out of the 7 strains of pHyg/TauT-transferred cell showed antibody yields higher than the highest yield in pHyg-transferred cell.

FIG. 22 shows anti-glypican-3 antibody yield plots on day 7 of 50 ml shaker flask fed-batch culture. The antibody yield in pHyg/TauT-transferred cell was superior to that in pHyg-transferred cell.

FIG. 23 is a graph showing the survival ratio of a pHyg/TauT-transferred cell T10 (which showed high growth ability) in 1 L jar fed-batch culture. The survival ratio of T10 was 80% or more even on day 32 of the culture.

FIG. 24 is a graph showing the antibody yield of a pHyg/TauT-transferred cell T10 (which showed high growth ability during the expansion process in static culture) in 1 L jar fed-batch culture. The anti-glypican-3 antibody yield of T10 was 2.9 g/L on day 35 of the culture.

FIG. 25 shows the results of flow cytometric analysis indicating that TauT-transferred T10 cell is expressing TauT molecules on its cell membrane.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinbelow, embodiments of the present invention will be described in more detail.

The present invention provides a method of producing a polypeptide, comprising culturing a cell which strongly expresses cysteine sulfinic acid decarboxylase and has a transferred DNA encoding a desired polypeptide and thereby allowing the cell to produce the polypeptide.

In the method of the present invention, the cell may be either a natural cell capable of producing the desired polypeptide or a transformed cell into which a DNA encoding the desired polypeptide has been transferred. Preferably, a transformed cell into which a DNA encoding the desired polypeptide has been transferred is used.

In the method of the present invention, the desired polypeptide is not particularly limited. The polypeptide may be any polypeptide such as an antibody (e.g., anti-IL-6 receptor antibody, anti-IL-6 antibody, anti-glypican-3 antibody, anti-CD3 antibody, anti-CD20 antibody, anti-GPIIb/IIIa antibody, anti-TNF antibody, anti-CD25 antibody, anti-EGFR antibody, anti-Her2/neu antibody, anti-RSV antibody, anti-CD33 antibody, anti-CD52 antibody, anti-IgE antibody, anti-CD11a antibody, anti-VEGF antibody, anti-VLA4 antibody, and the like) or a physiologically active protein (e.g., granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), erythropoietin, interferon, interleukin such as IL-1 or IL-6, t-PA, urokinase, serum albumin, blood coagulation factor, PTH, and the like). An antibody is particularly preferred, and may be any antibody such as a natural antibody, a low molecular sized antibody (e.g., Fab, scFv, sc(Fv)2), a chimeric antibody, a humanized antibody, etc.

By using a cell which strongly expresses cysteine sulfinic acid decarboxylase, the yield of a polypeptide in the cell can be increased.

Cysteine sulfinic acid decarboxylase is originally known as an enzyme that converts alanine-3-sulfinic acid to hypotaurine. If cysteine sulfinic acid decarboxylase is strongly expressed in a CHO cell, the cell synthesizes an excess amount of β-alanine.

A cell which strongly expresses cysteine sulfinic acid decarboxylase is not particularly limited as long as the cell has an increased expression level of cysteine sulfinic acid decarboxylase compared to a corresponding natural cell. The natural cell is not particularly limited. A cell which is used as a host in the production of a recombinant protein (e.g., CHO cells) may be used.

As a cell which strongly expresses cysteine sulfinic acid decarboxylase, a cell into which a cysteine sulfinic acid decarboxylase gene has been artificially transferred may be given. A cell into which a cysteine sulfinic acid decarboxylase gene has been artificially transferred can be prepared by methods known to those skilled in the art. For example, such a cell may be prepared by incorporating a cysteine sulfinic acid decarboxylase gene into a vector and transforming the vector into a cell. Furthermore, the concept of “cells into which a cysteine sulfinic acid decarboxylase gene has been artificially transferred” encompasses herein cells in which an endogenous cysteine sulfinic acid decarboxylase gene has been activated by gene activation technology (see, for example, International Publication WO94/12650) so that cysteine sulfinic acid decarboxylase is strongly expressed.

As cysteine sulfinic acid decarboxylase to be strongly expressed in a cell, cysteine sulfinic acid decarboxylase derived from any organism may be used. Specifically, cysteine sulfinic acid decarboxylase derived from human, a rodent (such as mouse, rat or hamster), a puffer (such as Tiger puffer) or a sea squirt (such as Ciona intestnalis) may be used. Preferably, cysteine sulfinic acid decarboxylase derived from human, a rodent or the same species as the host cell may be used. For example, when the cell which is allowed to strongly express cysteine sulfinic acid decarboxylase is Chinese hamster ovary cells (CHO cells), the cysteine sulfinic acid decarboxylase is preferably derived from human or hamster.

Further, as a cysteine sulfinic acid decarboxylase gene to be strongly expressed in a cell, any one of the following DNAs (a) to (e) may be used.

(a) a DNA encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO: 2, 4, 6 or 8; (b) a DNA encoding a polypeptide which has an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 2, 4, 6 or 8 by substitution, deletion, addition and/or insertion of one or more amino acid residues and yet has cysteine sulfinic acid decarboxylase activity; (c) a DNA encoding a polypeptide having 70% or more amino acid sequence homology with the amino acid sequence as shown in SEQ ID NO: 2, 4, 6 or 8 and yet having cysteine sulfinic acid decarboxylase activity; (d) a DNA having the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5 or 7; (e) a DNA which hybridizes to a DNA complementary to a DNA having the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5 or 7 under stringent conditions and yet encodes a polypeptide having cysteine sulfinic acid decarboxylase activity.

The cell which strongly expresses cysteine sulfinic acid decarboxylase may be any cell, for example, eukaryotic cell such as animal, plant and yeast cells, prokaryotic cell such as E. coli and B. subtilis, etc. Preferably, animal cells such as CHO and COS cells are used, CHO cells are particularly preferred. In order to prepare a desired polypeptide, cells suitable for transfer of a gene encoding the desired polypeptide such as CHO-dhfr-cells are preferred.

Preferably, the cell of the present invention which strongly expresses cysteine sulfinic acid decarboxylase further expresses a taurine transporter strongly in order to prepare a desired polypeptide. By transferring a gene encoding the desired polypeptide into the cell and culturing the resultant cell in a medium, the desired polypeptide can be produced in a greater amount.

When a desired polypeptide is produced using a cell into which a cysteine sulfinic acid decarboxylase gene has been artificially transferred, the order of the transfer of a cysteine sulfinic acid decarboxylase gene and the transfer of a gene encoding a desired polypeptide is not particularly limited. A gene encoding a desired polypeptide may be transferred after the transfer of a cysteine sulfinic acid decarboxylase gene. Alternatively, a cysteine sulfinic acid decarboxylase gene may be transferred after the transfer of a gene encoding a desired polypeptide. It is also possible to transfer a cysteine sulfinic acid decarboxylase gene and a gene encoding a desired polypeptide simultaneously.

A cysteine sulfinic acid decarboxylase gene and a gene encoding a desired polypeptide may be transferred simultaneously in a single vector. Alternatively, they may be transferred separately using a plurality of vectors.

By using a cell which strongly expresses cysteine sulfinic acid decarboxylase and a taurine transporter, an intracellular ammonia concentration can decline.

It is known that taurine transporter is a membrane protein having the osmoregulatory function of taking up amino acids (such as taurine and β-alanine) into cells.

A cell which strongly expresses a taurine transporter is not particularly limited as long as the cell has an increased expression level of a taurine transporter compared to a corresponding natural cell. The natural cell is not particularly limited. A cell which is used as a host in the production of a recombinant protein (e.g., CHO cells) may be used.

As a cell which strongly expresses a taurine transporter, a cell into which a taurine transporter gene has been artificially transferred may be given. A cell into which a taurine transporter gene has been artificially transferred can be prepared by methods known to those skilled in the art. For example, such a cell may be prepared by incorporating a taurine transporter gene into a vector and transforming the vector into a cell.

As a taurine transporter to be strongly expressed in a cell, a taurine transporter derived from any organism may be used. Specifically, a taurine transporter derived from human or a rodent (such as mouse, rat or hamster) may be used. Preferably, a taurine transporter derived from human, a rodent or the same species as the host cell may be used. For example, when the cell which is allowed to strongly express a taurine transporter is Chinese hamster ovary cells (CHO cells), the taurine transporter is preferably derived from human or hamster.

Further, as a taurine transporter gene to be strongly expressed in a cell, any one of the following DNAs (a) to (e) encoding a taurine transporter may be used.

(a) a DNA encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO: 20, 22, 24 or 26; (b) a DNA encoding a polypeptide which has an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 20, 22, 24 or 26 by substitution, deletion, addition and/or insertion of one or more amino acid residues and yet has taurine transporter activity; (c) a DNA encoding a polypeptide having 70% or more amino acid sequence homology with the amino acid sequence as shown in SEQ ID NO: 20, 22, 24 or 26 and yet having taurine transporter activity; (d) a DNA having the nucleotide sequence as shown in SEQ ID NO: 19, 21, 23 or 25; (e) a DNA which hybridizes to a DNA complementary to a DNA having the nucleotide sequence as shown in SEQ ID NO: 19, 21, 23 or 25 under stringent conditions and yet encodes a polypeptide having taurine transporter activity.

Production of a desired polypeptide may be performed by transferring a gene encoding the desired polypeptide into a cell which strongly expresses a taurine transporter gene and a cysteine sulfinic acid decarboxylase gene and culturing the resultant cell in a medium. Furthermore, a desired polypeptide can be prepared by using a cell in which an endogenous gene has been activated by gene activation technology (see, for example, International Publication WO94/12650) so that a desired polypeptide has been produced.

When a desired polypeptide is produced using a cell into which a taurine transporter gene and a cysteine sulfinic acid decarboxylase gene have been artificially transferred, the order of the transfer of a taurine transporter gene, the transfer of a cysteine sulfinic acid decarboxylase gene and the transfer of a gene encoding a desired polypeptide is not particularly limited. A gene encoding a desired polypeptide may be transferred after the transfer of a taurine transporter gene and a cysteine sulfinic acid decarboxylase gene. Alternatively, a taurine transporter gene and a cysteine sulfinic acid decarboxylase gene may be transferred after the transfer of a gene encoding a desired polypeptide. It is also possible to transfer a taurine transporter gene, a cysteine sulfinic acid decarboxylase gene and a gene encoding a desired polypeptide simultaneously.

A taurine transporter gene, a cysteine sulfinic acid decarboxylase gene and a gene encoding a desired polypeptide may be transferred simultaneously in a single vector. Alternatively, they may be transferred separately using a plurality of vectors.

For culturing the cell which strongly expresses cysteine sulfinic acid decarboxylase (and which may strongly express a taurine transporter), media used in conventional cell culture (preferably, animal cell culture) may be used. These media usually contain amino acids, vitamins, lipid factors, energy sources, osmotic regulators, iron sources and pH regulators. The contents of these components are usually as follows: amino acids 0.05-1500 mg/L, vitamins 0.001-10 mg/L, lipid factors 0-200 mg/L, energy sources 1-20 g/L, osmotic regulators 0.1-10000 mg/L, iron sources 0.1-500 mg/L, pH regulators 1-10000 mg/L, trace metal elements 0.00001-200 mg/L, surfactants 0-5000 mg/L, growth cofactors 0.05-10000 μg/L and nucleosides 0.001-50 mg/L. However, the contents are not limited to these ranges and may be appropriately selected depending on the type of the cell to be cultured, the type of the desired polypeptide, and so on.

In addition to these components, trace metal elements, surfactants, growth cofactors, nucleosides, and the like may be added.

Specific examples of such components include amino acids, such as L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine, preferably, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine; vitamins, such as i-inositol, biotin, folic acid, lipoic acid, nicotinamide, nicotinic acid, p-aminobenzoic acid, calcium pantothenate, pyridoxal hydrochloride, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B₁₂ and ascorbic acid, preferably, biotin, folic acid, lipoic acid, nicotinamide, calcium pantothenate, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, vitamin B₁₂ and ascorbic acid; lipid factors, such as choline chloride, choline tartrate, linoleic acid, oleic acid and cholesterol, preferably, choline chloride; energy sources, such as glucose, galactose, mannose, and fructose, preferably, glucose; osmotic regulators, such as sodium chloride, potassium chloride, and potassium nitrate, preferably, sodium chloride; iron sources, such as iron EDTA, ferric citrate, ferrous chloride, ferric chloride, ferrous sulfate, ferric sulfate, and ferric nitrate, preferably, ferric chloride, iron EDTA, and ferric citrate; and pH regulators, such as sodium hydrogencarbonate, calcium chloride, sodium dihydrogen-phosphate, HEPES and MOPS, preferably, sodium hydrogencarbonate. Culture media containing any of these components may be given as examples.

Besides the above components, there may be added trace metal elements, such as copper sulfate, manganese sulfate, zinc sulfate, magnesium sulfate, nickel chloride, tin chloride, magnesium chloride and sodium subsilicate, preferably, copper sulfate, zinc sulfate and magnesium sulfate; surfactants, such as Tween 80 and Pluronic F68; growth cofactors, such as recombinant insulin, recombinant IGF-1, recombinant EGF, recombinant FGF, recombinant PDGF, recombinant TGF-α, ethanolamine hydrochloride, sodium selenite, retinoic acid and putrescine dihydrochloride, preferably, sodium selenite, ethanolamine hydrochloride, recombinant IGF-1 and putrescine dihydrochloride; and nucleosides, such as deoxyadenosine, deoxycytidine, deoxyguanosine, adenosine, cytidine, guanosine and uridine. In preferable examples of above media, antibiotics, such as streptomycin, penicillin-G potassium and gentamicin, and pH-indicators, such as Phenol Red, may be contained.

The pH of the medium varies depending on the cell to be cultured. Generally, pH 6.8-7.6 is appropriate. In many cases, pH 7.0-7.4 is appropriate.

It is also possible to use a commercial medium for animal cell culture, e.g., D-MEM (Dulbecco's Modified Eagle Medium), D-MEM/F-12 1:1 Mixture (Dulbecco's Modified Eagle Medium Nutrient Mixture F-12), RPMI1640, CHO-S-SFMII (Invitrogen), CHO-SF (Sigma-Aldrich), EX-CELL 301 (JRH Biosciences), CD-CHO (Invitrogen), IS CHO-V (Irvine Scientific), PF-ACF-CHO (Sigma-Aldrich) or the like.

Alternatively, the medium may be a serum-free medium.

When the cell which strongly expresses cysteine sulfinic acid decarboxylase (and which may strongly express a taurine transporter) is CHO cells, CHO cells may be cultured by methods known to those skilled in the art. For example, CHO cells may be cultured usually in an atmosphere with a CO₂ concentration in the gas phase of 0 to 40%, preferably 2 to 10%, at 30 to 39° C., preferably about 37° C.

As is clear from the Examples described later, production of waste products (such as lactate) which turn to be cell growth inhibitory substances can be inhibited in a cell strongly expressing cysteine sulfinic acid decarboxylase. As a result, the cell shows the effect of maintaining a high survival ratio. The cell of the present invention is capable of culturing for twenty days or a longer period, preferably thirty days or a longer period, more preferably two months or a still longer period.

Further, when a desired polypeptide, such as an antibody, is produced in cultured cells, the cells come into a highly concentrated state (about 1×10⁷ cells/ml) at the late-stage of culture, and the influence of waste products such as lactate becomes extremely high. When a desired polypeptide is produced using the cell which strongly expresses cysteine sulfinic acid decarboxylase, a high survival ratio is maintained even at the late-stage of culture, and an improvement can be expected in the yield of the desired polypeptide.

An appropriate culture period for producing a desired polypeptide using the cell which strongly expresses cysteine sulfinic acid decarboxylase is usually 1 day to 3 months, preferably 1 day to 2 months, more preferably 1 day to 1 month.

With respect to various culture devices for animal cell culture, a fermentor type tank culture device, an air lift type culture device, a culture flask type culture device, a spinner flask type culture device, a microcarrier type culture device, a fluidized bed type culture device, a hollow fiber type culture device, a roller bottle type culture device, a packed bed type culture device, or the like may be used.

Culture may be performed by any culture method such as batch culture, fed-batch culture or continuous culture. Preferably, fed-batch culture or continuous culture is used. Fed-batch culture is more preferred.

When the cell which strongly expresses cysteine sulfinic acid decarboxylase (and which may strongly express a taurine transporter) is cultured, taurine may be added to the medium in order to promote taurine uptake into cells.

The present invention provides a novel polypeptide which is any one of the following (A) to (E), provided that a polypeptide having the amino acid sequence as shown in SEQ ID NO: 4, a polypeptide having the amino acid sequence as shown in SEQ ID NO: 6, and a polypeptide having the amino acid sequence as shown in SEQ ID NO: 8 are excluded:

(A) a polypeptide having the amino acid sequence as shown in SEQ ID NO: 2; (B) a polypeptide which has an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 2 by substitution, deletion, addition and/or insertion of one or more amino acid residues and yet has cysteine sulfinic acid decarboxylase activity; (C) a polypeptide having 97% or more amino acid sequence homology with the amino acid sequence as shown in SEQ ID NO: 2 and yet having cysteine sulfinic acid decarboxylase activity; (D) a polypeptide encoded by a DNA having the nucleotide sequence as shown in SEQ ID NO: 1; or (E) a polypeptide encoded by a DNA which hybridizes to a DNA complementary to a DNA having the nucleotide sequence as shown in SEQ ID NO: 1 under stringent conditions and yet encodes a polypeptide having cysteine sulfinic acid decarboxylase activity.

The novel polypeptides of the present invention are hamster cysteine sulfinic acid decarboxylase and those polypeptides which are functionally equivalent thereto. In the present invention, the expression “a polypeptide that is functionally equivalent to cysteine sulfinic acid decarboxylase” is used to mean that the polypeptide has a decarboxylation activity that is equivalent to the activity possessed by hamster cysteine sulfinic acid decarboxylase, such as enzyme activity for synthesizing hypotaurine from 3-sulfinic acid alanine, enzyme activity for synthesizing taurine from cysteic acid, and enzyme activity as of glutamate decarboxylase for synthesizing β-alanine from aspartic acid. Such a polypeptide encompasses, for example, mutants of hamster cysteine sulfinic acid decarboxylase.

As methods well-known to those skilled in the art for preparing polypeptides functionally equivalent to a specific polypeptide, methods of introducing mutations into polypeptides may be given. For example, those skilled in the art could prepare polypeptides functionally equivalent to hamster cysteine sulfinic acid decarboxylase by appropriately introducing mutations into amino acids of hamster cysteine sulfinic acid decarboxylase by site-directed mutagenesis (Hashimoto-Gotoh, T. et al. (1995) Gene 152, 271-275; Zoller, M J, and Smith, M. (1983) Methods Enzymol. 100, 468-500; Kramer, W. et al. (1984) Nucleic Acids Res. 12, 9441-9456; Kramer W, and Fritz H J (1987) Methods. Enzymol. 154, 350-367; Kunkel, T A (1985) Proc Natl Acad Sci USA. 82, 488-492; Kunkel (1988) Methods Enzymol. 85, 2763-2766). Mutations in amino acids may also occur in nature. Thus, a polypeptide which has an amino acid derived from the amino acid sequence of the hamster cysteine sulfinic acid decarboxylase of the present invention by mutation of one or more amino acids and is functionally equivalent to hamster cysteine sulfinic acid decarboxylase is also included in the novel polypeptide of the present invention.

Specific examples of polypeptides functionally equivalent to the hamster cysteine sulfinic acid decarboxylase of the present invention include, but are not limited to, a polypeptide having an amino acid sequence derived from the amino acid sequence of the hamster cysteine sulfinic acid decarboxylase by deletion of one or more amino acids, preferably 1-30 amino acids, more preferably 1-10 amino acids; a polypeptide having an amino acid sequence derived from the amino acid sequence of the hamster cysteine sulfinic acid decarboxylase by addition of one or more amino acids, preferably 1-30 amino acids, more preferably 1-10 amino acids; and a polypeptide having an amino acid sequence derived from the amino acid sequence of the hamster cysteine sulfinic acid decarboxylase by substitution of one or more amino acids, preferably 1-30 amino acids, more preferably 1-10 amino acids, with other amino acids.

Amino acid residues to be mutated are not particularly limited. Preferably, amino acid residues are mutated to other amino acids in which the nature of the initial amino acid side chain is conserved. Specific examples of the nature of amino acid side chain include hydrophobic amino acids (A, I, L, M, F, P, W, Y and V), hydrophilic amino acids (R, D, N, C, E, Q, G, K, S and T), amino acids with an aliphatic side chain (G, A, V, L, I and P), amino acids with a hydroxyl group-containing side chain (S, T and Y), amino acids with a sulfur atom-containing side chain (C and M), amino acids with a carboxylic acid and amide-containing side chain (D, N, E and Q), amino acids with a base-containing side chain (R, K and H) and amino acids with an aromatic-containing side chain (H, F, Y and W) (In parentheses are one-letter codes for amino acids).

It has been reported that a polypeptide having an amino acid sequence derived from an original amino acid sequence by modification (such as deletion, addition and/or substitution of one or more amino acids) maintains the biological activity of the original polypeptide (Mark, D. F. et al., Proc. Natl. Acad. Sci. USA (1984) 81, 5662-5666; Zoller, M. J. & Smith, M. Nucleic Acids Research (1982) 10, 6487-6500; Wang, A. et al., Science 224, 1431-1433; Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409-6413).

As one example of the polypeptide in which one or more amino acid residues are added to the hamster cysteine sulfinic acid decarboxylase of the present invention, a fusion polypeptide comprising the hamster cysteine sulfinic acid decarboxylase may be given. Such a fusion polypeptide is composed of the protein of the invention (hamster cysteine sulfinic acid decarboxylase) and other polypeptide fused thereto. Such a fusion polypeptide is included in the present invention. Such a fusion polypeptide may be prepared by linking a gene encoding the hamster cysteine sulfinic acid decarboxylase of the present invention in frame with a gene encoding the other polypeptide, transferring the resultant DNA into an expression vector and expressing the DNA in a host cell. Techniques known to those skilled in the art may be used. There is no limitation on the polypeptide to be fused to the polypeptide of the present invention.

Examples of polypeptides to be fused to the polypeptide of the present invention include, but are not limited to, FLAG (Hopp, T. P. et al., BioTechnology (1988) 6, 1204-1210), 6×His comprising six histidine (His) residues, 10×His, influenza hemagglutinin (HA), human c-myc fragment, VSV-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, α-tubulin fragment, B-tag, protein C fragment, glutathione-S-transferase (GST), influenza hemagglutinin (HA), immunoglobulin constant region, β-galactosidase and maltose-binding protein (MBP).

A commercially available gene encoding such polypeptide is fused to the gene encoding the polypeptide of the present invention. The fused gene thus prepared is expressed to prepare a fused polypeptide.

An alternative method known to those skilled in the art for preparing polypeptides functionally equivalent to a specific polypeptide is a method using the hybridization technique (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. Press, 1989). Those skilled in the art could routinely isolate a DNA highly homologous to the DNA sequence of the hamster cysteine sulfinic acid decarboxylase of the present invention based on that DNA sequence or a part thereof, and isolate polypeptides functionally equivalent to the hamster cysteine sulfinic acid decarboxylase from that DNA. Thus, a polypeptide which is encoded by a DNA hybridizing to the DNA, or a part thereof, encoding the hamster cysteine sulfinic acid decarboxylase of the present invention and is functionally equivalent to the hamster cysteine sulfinic acid decarboxylase of the present invention is also included in the polypeptide of the present invention.

Hybridization conditions for isolating a DNA encoding a polypeptide functionally equivalent to the hamster cysteine sulfinic acid decarboxylase of the present invention can be appropriately selected by those skilled in the art. For example, low stringent hybridization conditions may be given. Low stringent hybridization conditions are, for example, 42° C., 2×SSC and 0.1% SDS, preferably 50° C., 2×SSC and 0.1% SDS. More preferably, high stringent conditions may be given. For example, high stringent conditions are 65° C., 2×SSC and 0.1% SDS. Under these conditions, as the hybridization temperature is lowered, not only DNAs with high homology but also DNAs with only low homology are obtained. Conversely, it is expected that only those DNAs with high homology are obtained as the hybridization temperature is elevated. However, not only the temperature but also a plurality of factors (such as salt concentrations) affect the stringency of hybridization. Those skilled in the art could appropriately select these factors to realize similar stringency.

The polypeptide encoded by a DNA isolated by these hybridization techniques usually has high homology with the hamster cysteine sulfinic acid decarboxylase of the present invention in the amino acid sequence. The polypeptide of the present invention also include those polypeptides which are functionally equivalent to the hamster cysteine sulfinic acid decarboxylase of the present invention and have high homology with the amino acid sequence of the hamster cysteine sulfinic acid decarboxylase of the present invention. The term “high homology” refers to usually 97% or more homology, preferably 98% or more homology, more preferably 99% or more homology. For determination of the homology of polypeptides, the algorithm described in Wilbur, W. J. and Lipman, D. J., Proc. Natl. Acad. Sci. USA (1983) 80, 726-730 may be followed.

The polypeptide of the present invention may vary in amino acid sequence, molecular weight, isoelectric point, presence or absence of sugar chains, morphology, etc. depending on the cell or host that produce the polypeptide or the purification method that will be described later. However, as long as the resultant polypeptide has functions equivalent to the functions of the hamster cysteine sulfinic acid decarboxylase of the present invention, the polypeptide is included in the present invention. For example, when the polypeptide of the present invention is expressed in a prokaryote (e.g., Escherichia coli), a methionine reside is added to the N-terminus of the initial amino acid sequence of the polypeptide. When the polypeptide of the present invention is expressed in a eukaryote (e.g., a mammalian cell), the N-terminal signal sequence is removed. The polypeptide of the present invention includes such polypeptides.

The polypeptide of the present invention may be prepared as a recombinant polypeptide or a natural polypeptide by methods known to those skilled in the art. A recombinant polypeptide may be prepared by incorporating a DNA encoding the polypeptide of the present invention in an appropriate expression vector, introducing the vector into an appropriate host cell, collecting the resultant transformant, extracting a crude polypeptide, and then purifying the polypeptide by chromatography (such as ion exchange, reversed phase or gel filtration chromatography, or affinity chromatography in which an antibody to the polypeptide of the present invention is fixed in a column) or a combination of these chromatographic techniques.

When the polypeptide of the present invention is expressed in a host cell (e.g., animal cell or E. coli) as a fusion polypeptide with glutathione-S-transferase polypeptide or as a recombinant polypeptide with histidine residues added thereto, the expressed polypeptide may be purified with a glutathione column or a nickel column.

After purification of a fusion polypeptide, regions other than the polypeptide of interest may be cut off by thrombin or factor Xa and removed from the fusion polypeptide.

When the polypeptide of the present invention is a natural polypeptide, the polypeptide may be isolated by purification methods known to those skilled in the art. For example, an extract from tissues or cells expressing the polypeptide of the present invention may be applied to an affinity column to which an antibody to the hamster cysteine sulfinic acid decarboxylase described later is bound. The antibody may be either a polyclonal antibody or a monoclonal antibody.

Further, the present invention provides a novel DNA encoding cysteine sulfinic acid decarboxylase, which is any one of the following (a) to (e), provided that DNA having the nucleotide sequence as shown in SEQ ID NO: 3, DNA having the nucleotide sequence as shown in SEQ ID NO: 5, and DNA having the nucleotide sequence as shown in SEQ ID NO: 7 are excluded:

(a) a DNA encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO: 2; (b) a DNA encoding a polypeptide which has an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 2 by substitution, deletion, addition and/or insertion of one or more (several) amino acid residues and yet has cysteine sulfinic acid decarboxylase activity; (c) a DNA encoding a polypeptide having 97% or more amino acid sequence homology with the amino acid sequence as shown in SEQ ID NO: 2 and yet having cysteine sulfinic acid decarboxylase activity; (d) a DNA having the nucleotide sequence as shown in SEQ ID NO: 1; or (e) a DNA which hybridizes to a DNA complementary to a DNA having the nucleotide sequence as shown in SEQ ID NO: 1 under stringent conditions and yet encodes a polypeptide having cysteine sulfinic acid decarboxylase activity.

The novel DNA of the present invention is used to prepare a cell which strongly expresses cysteine sulfinic acid decarboxylase and thereafter used in the in vivo or in vitro production of a desired polypeptide as described above. The novel DNA of the present invention may take any form as long as it is capable of encoding a polypeptide having cysteine sulfinic acid decarboxylase activity. That is, the DNA may be, for example, a cDNA synthesized from mRNA, a genomic DNA or a chemically synthesized DNA. It should be noted that, as long as the DNA is capable of encoding the polypeptide of the present invention, the DNA may have any nucleotide sequence based on the degeneracy of genetic codes.

The DNA of the present invention may be prepared by methods known to those skilled in the art. For example, the DNA may be prepared by preparing a cDNA library from a cell expressing a polypeptide having cysteine sulfinic acid decarboxylase activity and performing hybridization using a part of the DNA sequence of the present invention (e.g., SEQ ID NO: 1) as a probe. The cDNA library may be prepared, for example, by the method described in Sambrook, J. et al., Molecular Cloning, Cold Spring Harbor Laboratory Press (1989). Alternatively, a commercial cDNA library may be used. It is also possible to prepare the DNA of the present invention by preparing RNA from a cell expressing a polypeptide having cysteine sulfinic acid decarboxylase activity, synthesizing oligo DNA molecules based on the DNA sequence of the present invention (e.g., SEQ ID NO: 1), and performing PCR using the oligo DNA molecules as primers to thereby amplify a cDNA encoding cysteine sulfinic acid decarboxylase.

Further, by determining the nucleotide sequence of the resultant cDNA, it is possible to determine the translation region encoding the polypeptide and to obtain the amino acid sequence of the polypeptide of the present invention. Further, by screening a genomic library using the resultant cDNA as a probe, it is possible to isolate a genomic DNA.

Specifically, the following procedures may be used. First, mRNA is isolated from cells, tissues or the like expressing the polypeptide of the present invention. For the isolation of mRNA, the total RNA is prepared by known methods, for example, the guanidine ultracentrifugation method (Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294-5299), the AGPC method (Chomczynski, P. and Sacchi, N., Anal. Biochem. (1987) 162, 156-159) or the like, and then mRNA is purified from the total RNA using mRNA Purification Kit (Pharmacia), etc. Alternatively, mRNA may be prepared directly using QuickPrep mRNA Purification Kit (Pharmacia).

From the resultant mRNA, cDNA is synthesized using a reverse transcriptase. Alternatively, cDNA may be synthesized using a kit such as AMV Reverse Transcriptase First-Strand cDNA Synthesis Kit (SEIKAGAKU CORPORATION). It is also possible to synthesize and amplify cDNA according to the 5′-RACE method (Frohman, M. A. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002; Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932) using 5′-Ampli FINDER RACE Kit (Clontech) and polymerase chain reaction (PCR) with primers.

A DNA fragment of interest is prepared from the resultant PCR product and ligated to a vector DNA to thereby prepare a recombinant vector. The vector is introduced into a host (e.g., E. coli), followed by selection of resultant colonies to thereby obtain a desired recombinant vector. The nucleotide sequence of the DNA of interest may be confirmed by a known method such as the dideoxynucleotide chain termination method.

Further, a nucleotide sequence of a higher expression efficiency can be designed for the DNA of the present invention by considering the frequency of codon usage in the host to be used for expression (Grantham, R. et al., Nucleic Acids Research (1981) 9, p. 43-74). Further, the DNA of the present invention can be modified using commercially available kits or known methods. Examples of such modifications include, but are not limited to, digestion with restriction enzymes, insertion of synthetic oligonucleotides or appropriate DNA fragments, addition of linkers, and insertion of an initiation codon (ATG) and/or a termination codon (TAA, TGA or TAG).

The DNA of the present invention also includes a DNA which hybridizes to a DNA having the nucleotide sequence as shown in SEQ ID NO: 1 under stringent conditions and encodes a polypeptide functionally equivalent to cysteine sulfinic acid decarboxylase.

Stringent conditions can be appropriately selected by those skilled in the art, including, for example, low stringent conditions. Low stringent conditions refer to, for example, 42° C., 2×SSC and 0.1% SDS, preferably 50° C., 2×SSC and 0.1% SDS. More preferably, high stringent conditions may be selected. High stringent conditions refer to, for example, 65° C., 2×SSC and 0.1% SDS. Under these conditions, as the hybridization temperature is elevated, DNAs with a higher homology can be obtained. The above-described DNA which hybridizes is preferably a DNA derived from nature, e.g., cDNA or chromosomal DNA.

These DNAs isolated by hybridization techniques usually have a high nucleotide sequence identity with a DNA encoding the hamster cysteine sulfinic acid decarboxylase of the present invention. The DNA of the present invention also includes a DNA which encodes a polypeptide functionally equivalent to the hamster cysteine sulfinic acid decarboxylase of the present invention and has high identity with a DNA encoding the hamster cysteine sulfinic acid decarboxylase of the present invention. The term “high identity” refers to usually 96% or more homology, preferably 98% or more homology, more preferably 99% or more identity. The identity of nucleotide sequences may be determined by algorithm BLAST (Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Based on this algorithm, programs such as BLASTN and BLASTX have been developed (Altschul et al. J. Mol. Biol. 215:403-410, 1990). When nucleotide sequences are analyzed by BLASTN based on BLAST, parameters may be set as score=100 and wordlength=12, for example. Specific procedures for these analysis methods are known (http://www.ncbi.nlm.nih.gov.).

Further, the present invention provides a vector into which the DNA of the present invention has been inserted. The vector of the present invention is useful for retaining the DNA of the present invention within the host cell and for permitting expression of the polypeptide of the present invention (i.e., hamster cysteine sulfinic acid decarboxylase or a polypeptide functionally equivalent thereto). The vector of the present invention is also useful for permitting the host cell to strongly express the cysteine sulfinic acid decarboxylase. By permitting the host cell to strongly express the cysteine sulfinic acid decarboxylase, a production of a desired polypeptide in the host cell can be increased.

When the host cell to be used is E. coli, it is preferable that the vector has a replication origin (“ori”) so that the vector is largely amplified in E. coli (e.g., JM109, DH5α, HB101 and XL1-Blue) and prepared in large quantity, and also genes for selecting transformed E. coli (e.g., drug resistance genes that enable discrimination of transformant with some drugs such as ampicillin, tetracycline, kanamycin or chloramphenicol). Examples of preferable vectors include, but are not limited to, M13 vectors, pUC vectors, pBR322, pBluescript and pCR-Script. In addition to these vectors, pGEM-T, pDIRECT, pT7, etc. may be enumerated when the vector is used for the purpose of subcloning a cDNA and cutting off the subcloned cDNA. When the vector is used for the purpose of producing the polypeptide of the present invention, an expression vector is especially useful. When expression in E. coli is intended, the expression vector preferably has the above-described features so that the vector is amplified in E. coli, and it also preferably has a promoter which allows efficient expression in E. coli such as JM109, DH5α, HB101 or XL1-Blue, e.g., lacZ promoter (Ward et al, Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427), araB promoter (Better et al, Science (1988) 240, 1041-1043) or T7 promoter. Specific examples of such vector include, in addition to those listed above, pGEX-5X-1 (Pharmacia), QIAexpress system (Qiagen), pEGFP, or pET (for its host, T7 RNA polymerase-expressing BL21 is preferred).

The vector may comprise signal sequences for polypeptide secretion. When the polypeptide is to be produced in the periplasm of E. coli, pelB signal sequence (Lei, S. P. et al., J. Bacteriol. (1987) 169, 4379) may be used as a signal sequence for polypeptide secretion. Introduction of the vector into a host cell may be performed, for example, by the calcium chloride method or electroporation.

In cases where a host cell other than E. coli is used, vectors useful for producing the polypeptide of the present invention include, but are not limited to, mammal-derived expression vectors [e.g., pcDNA3 from Invitrogen; pEGF-BOS (Nucleic Acids. Res. 1990, 18(17), p. 5322); pEF, pCDM8], insect cell-derived expression vectors (e.g., Bac-to-BAC baculovirus expression system from GIBCO BRL; pBacPAK8), plant-derived expression vectors (e.g., pMH1, pMH2), animal virus-derived expression vectors (e.g., pHSV, pMV, pAdexLcw), retrovirus-derived expression vectors (e.g., pZIpneo), yeast-derived expression vectors (e.g., Pichia Expression Kit from Invitrogen; pNV11; SP-Q01), and Bacillus subtilis-derived expression vectors (e.g., pPL608, pKTH50).

When expression of the polypeptide in animal cells (such as CHO cells, COS cells, NIH3T3 cells, etc.) is intended, the vector preferably has a promoter necessary for expressing the polypeptide in those cells. Examples of such promoter include, but are not limited to, SV40 promoter (Mulligan et al, Nature (1979) 277, 108), MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322) and CMV promoter. More preferably, the vector also has genes for selecting transformed cells (e.g., drug resistance genes that enable discrimination with drugs such as neomycin or G418). Examples of vectors having such properties include, but are not limited to, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13.

Further, when stable expression of a gene of interest and intracellular amplification of the copy number of the gene are indented, the following method may be used. Briefly, into CHO cells lacking a nucleic acid synthesis pathway, a vector having DHFR gene that complements the lack (e.g., pCHOI) is introduced, followed by amplification with methotrexate (MTX). On the other hand, when tentative expression of a gene of interest is intended, a method may be used in which COS cells carrying a gene expressing SV40T antigen on the chromosome is transformed with a vector having the replication origin of SV40 (e.g., pcD). As the replication origin, a replication origin derived from polyomavirus, adenovirus or bovine papillomavirus (BPV) may also be used. Further, the expression vector may contain selectable markers for amplifying the copy number of the gene in a host cell system. Examples of such selectable markers include, but are not limited to, aminoglycoside phosphotransferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene and dihydrofolate reductase (dhfr) gene.

The present invention also provides a host cell into which the DNA of the present invention (which may be incorporated into a vector) has been transferred. The host cell into which the DNA of the present invention (which may be incorporated into a vector) is transferred is not particularly limited. For example, E. coli or various animal cells may be used. The host cell of the present invention may be used, for example, as a production system for the preparation or expression of the polypeptide of the present invention. If DNA encoding a desired polypeptide is transferred into the host cell of the present invention, the host cell of the present invention can strongly express cysteine sulfinic acid decarboxylase, which leads to an increased production of the desired polypeptide. DNA encoding a taurine transporter (which may be incorporated into a vector) may be further transferred into the host cell of the present invention. By transferring DNA encoding a desired polypeptide and DNA encoding a taurine transporter into the host cell of the present invention, the yield of the desired polypeptide can be increased. For the production of the polypeptide, there are in vivo and in vitro production systems. Examples of in vitro production systems include systems using eukaryotes and systems using prokaryotes.

The present invention also provides a cell into which DNA encoding cysteine sulfinic acid decarboxylase and DNA encoding a taurine transporter have been transferred.

When eukaryotes are used, animal cells, plant cells, fungal cells, etc. may be used as the host. Specific examples of animal cells include mammalian cells, such as CHO cells (J. Exp. Med. (1995) 108, 945), COS cells, 3T3 cells, myeloma cells, BHK (baby hamster kidney) cells, HeLa cells and Vero cells; amphibian cells, such as oocytes of Xenopus laevis (Valle, et al., Nature (1981) 291, 358-340); or insect cells, such as sf9, sf21 and Tn5 cells. Among CHO cells, dhfr-CHO lacking DHFR gene (Proc. Natl. Acad. Sci. USA (1980) 77, 4216-4420) and CHO K-1 (Proc. Natl. Acad. Sci. USA (1968) 60, 1275) are used with particular advantage. When high expression is intended in an animal cell, CHO cells are especially preferred. Introduction of DNA (which may be incorporated into a vector) into the host cell may be performed by such methods as the calcium phosphate method, the DEAE dextran method, a method using a cationic ribosome DOTAP (Boehringer-Mannheim), electroporation, lipofection, etc.

As plant cells for polypeptide production, a Nicotiana tabacum-derived cell is known as a polypeptide production system and this may be subjected to callus culture. As fungal cells for polypeptide production, specific examples include yeast belonging to the genus Saccharomyces, e.g., Saccharomyces cerevisiae, and filamentous fungi belonging to the genus Aspergillus, e.g., Aspergillus niger.

When prokaryotes are used, production systems using bacterial cells are known. Specific examples of such bacterial cells include E. coli (such as JM109, DH5α, HB101) and Bacillus subtilis.

The polypeptide encoded by a gene of interest may be obtained by transforming these cells with the gene of interest and culturing the transformed cells in vitro. The culture may be performed by known methods. For example, as a culture broth for animal cells, a medium such as DMEM, MEM, RPMI1640 or IMDM may be used. A serum supplement such as fetal calf serum (FCS) may be used jointly. Alternatively, serum-free culture may be performed. The pH during culture is preferably about 6 to 8. The culture is usually performed at about 30-40° C. for about 15-200 hours. If necessary, replacement of the medium, aeration and agitation are carried out.

On the other hand, in vivo production systems include those using animals or plants. A gene of interest is transferred into these animals or plants to produce the polypeptide in the animal bodies or plant bodies. Then, the polypeptide is collected. The term “host” as used herein includes such animals or plants.

When animals are used, available production systems include those using mammals or insects. Goat, pig, sheep, mouse and cattle may be used as mammals (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). When mammals are used, transgenic animals may be used.

First, a gene of interest is fused to a gene encoding a polypeptide produced inherently in milk (such as goat β-casein) to thereby prepare a fusion gene. A DNA fragment containing this fusion gene is injected into a goat embryo, which is then implanted in the uterus of a female goat. The polypeptide of interest can be obtained from the milk produced by transgenic goats born from the goat which accepted the embryo or the offspring of the transgenic goats. In order to increase the yield of milk containing the polypeptide produced by the transgenic goats, hormones may be appropriately administered to the transgenic goats (Ebert, K. M. et al., Bio/Technology (1994) 12, 699-702).

Examples of insects which may be used include silkworm. In this case, silkworm is infected with baculovirus carrying a transferred gene encoding the polypeptide of interest. The polypeptide of interest can be obtained from the body fluid of the silkworm (Susumu, M. et al., Nature (1985) 315, 592-594).

Furthermore, when plants are used, tobacco can typically be used. When tobacco is used, a gene encoding the polypeptide of interest is inserted into a plant expression vector (e.g., pMON 530), which is then transferred into a bacterium such as Agrobacterium tumefaciens. A tobacco plant (e.g., Nicotiana tabacum) is infected with the resultant bacterium. The polypeptide of interest can be obtained from leaves of this plant (Julian, K.-C. Ma et al., Eur. J. Immunol. (1994) 24, 131-138).

The polypeptide thus obtained can be isolated from the inside of the host cell or from its outside (e.g., medium), and purified to a substantially pure and homogeneous polypeptide. Isolation and purification of polypeptides can be performed using conventional isolation and purification methods for polypeptides, and are not limited in any way. For example, polypeptides can be isolated and purified by appropriate selection and combination of various tools and techniques, such as chromatography columns, filters, ultrafiltration, salting-out, precipitation with solvent, extraction with solvent, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc.

Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, adsorption chromatography, etc. (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed. Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These chromatographic techniques can be carried out using liquid phase chromatography, for example, HPLC, FPLC, etc. The present invention also includes those polypeptides highly purified using these purification methods.

Before or after the purification, it is also possible to give optional modifications to the polypeptide or remove a partial peptide therefrom by reacting the polypeptide with an appropriate polypeptide modification enzyme. Examples of such enzyme include, but are not limited to, trypsin, chymotrypsin, lysyl endopeptidase, protein kinase and glucosidase.

The present invention also provides an antibody that binds to the polypeptide of the present invention. The form of the antibody of the present invention is not particularly limited; the antibody of the present invention includes polyclonal antibodies and monoclonal antibodies. In addition, antisera obtained by immunizing immune animals such as rabbit with the polypeptide of the present invention, and polyclonal antibodies and monoclonal antibodies of every class are also included in the antibody of the present invention.

The polypeptide used as a sensitizing antigen may be an intact polypeptide or a partial peptide thereof. Examples of such partial peptides include amino (N)-terminal fragments and carboxyl (C)-terminal fragments of the polypeptide. The term “antibody” as used herein means an antibody that reacts with the full length polypeptide or fragments thereof.

After inserting a gene encoding the polypeptide of the present invention or a fragment thereof into a known expression vector and transforming the host cell described earlier in this specification with this expression vector, the desired polypeptide or a fragment thereof is obtained from inside and outside of the host cells by a known method. This polypeptide or a fragment thereof may be used as a sensitizing antigen. Alternatively, cells expressing the polypeptide of the present invention or a lysate thereof, or a chemically synthesized polypeptide of the present invention may also be used as a sensitizing antigen.

Although there is no particular limitation on the species of mammals to be immunized with a sensitizing antigen, it is preferable to select mammals taking into consideration the compatibility with the parent cell to be used for cell fusion, and animals of the rodent, lagomorph and primate are generally used.

For example, mouse, rat, hamster and the like are used as rodent animals; rabbit is used as a lagomorphic animal, and monkey is used as a primate animal. Among monkeys, catarrhine monkeys (old world monkeys) are used, as exemplified by cynomolgus monkey (Macaca fascicularis), rhesus monkey, baboon, chimpanzee and the like.

Immunization of animals with a sensitizing antigen is carried out according to any known method. Generally, immunization is performed by intraperitoneal or subcutaneous injection of the antigen to mammals. Specifically, the antigen is appropriately diluted and suspended in PBS (Phosphate-Buffered Saline), physiological saline or the like, optionally mixed with a suitable amount of usual adjuvant (e.g., Freund's complete adjuvant), emulsified and then administered to mammals, preferably followed by several booster injections of the antigen mixed with an appropriate amount of Freund's incomplete adjuvant every 4 to 21 days. In addition, suitable carriers can be used at the time of immunization with the antigen. Subsequently, elevation of the level of a desired antibody in the sera of animals is confirmed by a conventional method.

In order to obtain polyclonal antibodies to the polypeptide of the present invention, the blood of mammals sensitized with the antigen is withdrawn after confirming elevation of the level of a desired antibody in sera. Then, sera are isolated from the blood by a known method. A serum containing a polyclonal antibody may be used as the polyclonal antibody. If necessary, a fraction containing the polyclonal antibody may be further isolated from the serum and used as the polyclonal antibody. For example, a fraction recognizing only the polypeptide of the present invention is obtained by using an affinity column to which the polypeptide of the present invention has been coupled, and further purified by using protein A or protein G column to prepare immunoglobulin G or M.

Monoclonal antibodies can be obtained by removing immunocytes from the mammals sensitized with the above-described antigen, after confirming the elevation of the level of a desired antibody in their sera, and then subjecting the immunocytes to cell fusion. Immunocytes preferably used for cell fusion are spleen cells. Parent cells to be fused to the above-described immunocytes are preferably mammalian myeloma cells, more preferably myeloma cells which have acquired characteristics for the selection of fused cells with drugs.

Cell fusion between the above-described immunocytes and myeloma cells can be carried out according to a known method, for example, the method of Milstein et al. (Galfre, G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46).

Hybridomas thus obtained by cell fusion are selected by culturing them in a conventional selection medium, for example, HAT culture medium (culture broth containing hypoxanthine, aminopterin and thymidine). Culturing in the HAT medium is continued for a sufficient time to kill other cells (non-fused cells) than desired hybridomas, usually for several days to several weeks. Then, conventional limiting culture-dilution method is performed to carry out screening and cloning of hybridomas producing the antibody of interest.

Subsequently, the resultant hybridomas are transplanted into the abdominal cavities of mice, and abdominal dropsies are collected from the mice to thereby obtain monoclonal antibodies. These monoclonal antibodies may be purified by ammonium sulfate precipitation, with protein A or protein G column, by DEAE ion exchange chromatography, with affinity column to which the polypeptide of the present invention has been coupled, etc. The antibody of the present invention can be used for purification and detection of the polypeptide of the present invention.

Further, the thus obtained monoclonal antibodies can also be prepared as recombinant antibodies using recombinant DNA techniques (see, for example, Borrebaeck, C. A. K. and Larrick, J. W., THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990). A recombinant antibody is produced by cloning a gene encoding the recombinant antibody from hybridomas or the antibody-producing immunocytes (such as sensitized lymphocytes), incorporating the gene into an appropriate vector, and transferring the vector into host cells for antibody production. The produced recombinant antibody is also included in the present invention.

The antibody of the present invention may be antibody fragments or modified antibodies as long as they are capable of binding to the polypeptide of the present invention. Examples of such antibody fragments include Fab, F(ab′)2, Fv, or a single-chain Fv (scFv) prepared by linking the Fv of H-chain to the Fv of L-chain via a suitable linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883). Specifically, antibody fragments are produced by digesting the antibody with enzymes, for example, papain or pepsin; or by constructing a gene encoding such a fragment, inserting the gene into an expression vector, and expressing it in a suitable host cell (see, for example, Co, M. S. et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, A. H., Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enymol. (1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. (1986) 121, 663-669; Bird, R. E. and Walker, B. W., Trends Biotechnol. (1991) 9, 132-137).

As a modified antibody, the antibody bound to various molecules such as polyethylene glycol (PEG) may be also used. The “antibody” of present invention also includes these modified antibodies. These modified antibodies can be prepared by chemically modifying the antibody obtained as described above. These modification methods have been already established in the art.

Antibodies obtained as described above can be purified to homogeneity. For the isolation and purification of the antibody used in present invention, any methods of isolation and purification used for conventional polypeptides may be used. For example, chromatography columns such as affinity chromatography columns, filters, ultrafiltration, salting-out, dialysis, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, etc. may be used independently or in appropriate combinations (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988) but these are not the sole examples. Concentration of the antibody obtained as described above can be determined by measuring absorbance or by a method such as enzyme-linked immunosorbent assay (ELISA).

Examples of columns used in affinity chromatography include protein A column and protein G column. As columns using protein A, Hyper D, POROS, Sepharose F. F. (Pharmacia), etc. may be given.

Examples of chromatography other than affinity chromatography include ion exchange chromatography, hydrophobic chromatography, gel filtration, reversed-phase chromatography, adsorption chromatography, etc. (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These chromatographic techniques can be carried out using liquid phase chromatography such as HPLC, FPLC, etc.

Furthermore, absorbance measurement, ELISA, EIA (enzyme immunoassay), MA (radioimmunoassay) or fluorescent antibody technique can be used as methods for measuring the antigen-binding activity of the antibody of present invention. When ELISA is used, the polypeptide of the present invention is added to plates on which the antibody of the present invention has been immobilized, and then a sample containing an antibody of interest (e.g., culture supernatant of antibody-producing cells or purified antibody) is added to the plates. A secondary antibody labeled with an enzyme (e.g., alkaline phosphatase) that recognizes the antibody is added to the plates. After incubating and washing the plates, a substrate for the enzyme (e.g., p-nitrophenyl phosphate) is added to determine the absorbance to thereby evaluate the antigen-binding activity. Instead of the entire polypeptide, a fragment thereof may be used. For example, fragments comprising its C-terminal or N-terminal may be used. For evaluating the activity of the antibody of the present invention, BIAcore (Pharmacia) may be used.

In one aspect, the present invention provides a method for producing a desired polypeptide, which is characterized in that cells capable of high-yield production of β-alanine and into which a desired polypeptide has been introduced, are cultured to increase the intracellular concentration of β-alanine in the cells during the culture, thereby improving production of the desired polypeptide.

The term “cells capable of high-yield production of β-alanine” is used herein to mean: cells in which the ratio of the intracellular concentration of β-alanine on the 6^(th) or subsequent days after the initiation of the culture to that at the initiation of the culture is 2 or greater, and preferably 5 or greater; or cells which are capable of increasing the β-alanine concentration in the culture medium on the 6^(th) or subsequent days after the initiation of the culture by 50 μM or more, and preferably by 100 μM or more.

As a method of measuring the ratio of two intracellular concentrations of β-alanine, the following method may be applied. Samples are collected at appropriate times from a culture medium containing cells in a 1-L jar feeding culture tank, such that the number of cells per sample is 50 to 500×10⁵, preferably 100 to 450×10⁵, and most preferably approximately 200×10⁵. The collected samples are centrifuged, and the obtained culture supernatants are isolated as samples for use in the subsequent measurement of amino acids in the culture medium. Thereafter, 1 ml of cooled sterile water having a protease inhibitor (Complete Mini; Roche Diagnostics; Protease inhibitor cocktail tablets) dissolved therein is added to the cell pellets, which are then pulsed on ice for 5 seconds using an ultrasonic cell disruptor (MISONIX ASTRASON MODEL XL2020), and such pulsing operation is then suspended for 5 seconds. This cycle is defined as one set of treatment, and a total of 12 sets of treatment are carried out, so as to completely disrupt the cells. The total amount of the solution after the treatment is applied to a centrifugal filtration unit to prepare filtrates with molecular weights of 5000 and less. The filtrates are used as samples for the measurement of intracellular amino acids. The absorbance at 570 nm of each sample is detected using a ninhydrin test solution-L 8500 set (Wako Pure Chemical Industries, Ltd.) and an improved model of a fully automatic amino acid analyzer (L-8500) manufactured by Hitachi, Ltd., and the obtained values are then compared with one another. Thus, the concentrations of various amino acids in the samples are determined. The concentration of β-alanine in the culture medium is directly measured. Thus, the determined concentrations are compared with one another on the μM order. Now consider the concentration of intracellular β-alanine. Since 1 mL of cooled sterile water has been added to the cell pellets, followed by ultrasonic cell disruption, the detection value of intracellular β-alanine derived from the CSAD strongly expressing strain at the initiation of the culture is defined as 1, and this detection value is compared with the detection values for each of the strongly expressing strains at the initiation of the culture, and on the 6^(th) and 12^(th) days after the initiation of the culture, so that each ratio is obtained. It is to be noted that the aforementioned culture is preferably carried out with 1 to 10×10⁵ cells/mL in an initial stage. In an exemplary method of measuring the concentration of β-alanine in a culture medium, culture may be carried out in a 1-L jar feeding culture tank, under the condition of approximately 2×10⁵ cells/mL in an initial stage and the concentration of β-alanine in the culture medium may be measured over time.

Alternatively, is another method of measuring the concentration of β-alanine in a culture medium, a culture supernatant may be measured using an amino acid analyzer, as described above.

In order to increase the amount of β-alanine produced in the cultured cells, a gene of a polypeptide having a function of accumulating intracellular β-alanine such as a protein having enzyme activity for synthesizing β-alanine from aspartic acid, may be expressed strongly, preferably by introducing DNA encoding the aforementioned gene into the cells.

Such proteins having enzyme activity for synthesizing β-alanine from aspartic acid include cysteine sulfinic acid decarboxylase, glutamate decarboxylase (GAD) (EC. 4.1.1.15), aspartate 1-decarboxylase (EC. 4.1.1.1), and the like.

Cells in which the intracellular concentration of β-alanine has been increased include cells into which DNA encoding cysteine sulfinic acid decarboxylase, glutamate decarboxylase (GAD), aspartate 1-decarboxylase (EC. 4.1.1.1), or a mutant thereof has been introduced.

A specific example of the cell of the present invention that are capable of high-yield production of β-alanine is cells in which the ratio of the concentration of intracellular β-alanine on the 6^(th) or subsequent days after the initiation of the culture to that at the initiation of the culture is 2 or greater, or the ratio of the concentration of intracellular β-alanine on the 12^(th) or subsequent days after the initiation of the culture to that at the initiation of the culture is 5 or greater, as measured by the same method as that described later in Examples.

Another specific example of the cells of the present invention that are capable of high-yield production of β-alanine is cells which are capable of increasing the β-alanine concentration in the culture medium on the 6^(th) or subsequent days after the initiation of the culture by 50 μM or more, and preferably by 100 μM or more, as measured by the same method as that described later in Examples.

In another aspect, the present invention provides a cultured cell in which the ratio of the intracellular concentration of β-alanine on the 6^(th) or subsequent days after the initiation of the culture to that at the initiation of the culture is 2 or greater.

In a further aspect, the present invention provides a cultured cell which is capable of increasing the β-alanine concentration in the culture medium on the 6^(th) or subsequent days after the initiation of the culture by 50 μM or more, and preferably by 100 μM or more.

In the present invention, the concept of “cells into which DNA has been transferred” encompasses not only cells into which exogenous DNA has been incorporated by genetic recombination technology; but also cells in which endogenous DNA has been activated by gene activation technology (see, for example, International Publication WO94/12650) so that expression of a protein corresponding to the endogenous DNA or transcription of the DNA has been initiated or increased.

EXAMPLES

Hereinbelow, the present invention will be described in more detail with reference to the following Examples. It should be noted that these Examples are provided only for illustrating the present invention and not for limiting the scope of the present invention.

Example 1 Cloning of CHO Cell-Derived Hamster Cysteine Sulfinic Acid Decarboxylase (CSAD) and Cysteine Dioxygenase, Type I (CDO1) Genes

Total RNA was extracted from anti-IL-6 receptor antibody-producing cells (A CHO DXB11 cell line into which an anti-IL-6 receptor antibody gene had been transferred) (Japanese Unexamined Patent Publication No. Hei 8-99902), and then cDNA was synthesized therefrom in a poly(A) dependent manner. Hamster CSAD and CDO1 genes were obtained by PCR using as a template the cDNA fragmented with three restriction enzymes, SalI, XhoI and EcoRI. As PCR primers, those containing the 5′-end and the 3′-end sequence conserved between rat and mouse CSADs or CDO1s were designed. The nucleotide sequences of the cloned genes were determined. From its homology with other CSAD or CDO1 genes of known species, the cloned gene was confirmed to encode hamster CASD (FIG. 1) or CDO1 (FIG. 2). The amino acid sequence of hamster CSAD has high homology with the known amino acid sequences of mouse CSAD (96% identity), rat CSAD (96% identity) and human CSAD (91% identity); it was predicted that hamster CSAD is an enzyme having the same activity. The amino acid sequence of hamster CDO1 has high homology with the known amino acid sequences of mouse CDO1 (99% identity), rat CDO1 (98% identity) and human CDO1 (93% identity); it was predicted that hamster CDO1 is an enzyme having the same activity. The nucleotide sequences of hamster, rat, mouse and human CSAD are shown in SEQ ID NOs: 1, 3, 5 and 7, respectively. The amino acid sequences of hamster, rat, mouse and human CSAD are shown in SEQ ID NOs: 2, 4, 6 and 8, respectively. The nucleotide sequences of hamster, rat, mouse and human CDO1 are shown in SEQ ID NOs: 9, 11, 13 and 15, respectively. The amino acid sequences of hamster, rat, mouse and human CDO1 are shown in SEQ ID NOs: 10, 12, 14 and 16, respectively. The nucleotide and amino acid sequences of bovine CDO1 are shown in SEQ ID NOs: 17 and 18, respectively.

Example 2 Increase in Antibody Yield, Inhibition of Lactate Production and Maintenance of Survival Ratio, as Caused by Transfer of Hamster CSAD

CMV promoter expression plasmid pHyg/CSAD (FIG. 3) or pHyg/CDO1 (FIG. 4) was constructed by adding Kozak sequence to the hamster CSAD (hereinafter, CSAD) or the hamster CDO1 (hereinafter, CDO1) gene obtained by cloning in Example 1. The plasmid pHyg/CSAD or pHyg/CDO1 was introduced by electroporation into the parent strain anti-glypican-3 antibody producing CHO cell (see WO 2006/006693). After selection of expression plasmid-transferred cells in the presence of hygromycin (200 μg/ml), all of the stably growing cell strains were expanded (pHyg/CSAD: 7 strains; pHyg/CDO1: 6 strains). The mRNA expression levels of CSAD and CDO1 were quantified by a TaqMan method. The two strains were confirmed to express mRNA at levels superior to that with the parent strain (in both CSAD and CDO1, the mRNA expression was 200 times or greater than that of the parent strain). The aforementioned cells (2×10⁵ cells/mL in an initial stage) were subjected to feeding culture in a 50-ml shaker. A comparison was made for the amount of an anti-glypican-3 antibody generated on the 14^(th) day at the late stage of the culture. The amount of the antibody produced in pHyg/CSAD-transferred cells was significantly higher than that in pHyg/CDO1-transferred cells (t-test: p<0.05) (FIG. 5). This result suggests that a strain capable of high-yield antibody production can be obtained by artificially expressing hamster CSAD. Subsequently, in anticipation of a synergistic effect that might be obtained by introduction of CSAD and TauT, pPur/CSAD (FIG. 6) was constructed and then introduced by electroporation into the pHyg/TauT-transferred cells (T10) prepared in Reference example 2. Selection was carried out in the presence of puromycin (6 pg/ml), and four stably growing strains were then amplified (pHyg/TauT+pPur/CSAD: TauT+CSAD-transferred strains). The mRNAs expression levels of TauT and CSAD in the 4 strains were quantified by a TaqMan method. As a result, strong expression of TauT and CSAD was confirmed in all the strains (expression of TauT mRNA in the 4 strains was almost at the same level as that of the parent TauT-transferred strain, and expression of CSAD mRNA in the 4 strains was 130 to 580 times higher than that of the TauT-transferred strain). The TauT-transferred strain was compared for culture profile with the TauT+CSAD-transferred strain by feeding culture of the cells in a 50-ml shaker (2×10⁵ cells/mL in an initial stage). As FIG. 7 shows, the average amount of an anti-glypican-3 antibody produced on the 14^(th) day at the late stage of culture in the TauT+CSAD-transferred strain (1343 mg/L) was higher than that in the TauT-transferred strain (1122 mg/L). Moreover, in terms of suppression of lactate production (FIG. 8) and retention of the survival ratio (FIG. 9) as well, the TauT+CSAD-transferred strain showed a higher potential. Furthermore, in order to confirm high potential in the amount of an antibody produced, the TauT+CSAD-transferred strain showing the highest survival ratio in FIG. 9 and the TauT-transferred strain were subjected to feeding culture in a 1-L jar (FIG. 10). A significant difference in the amount of an antibody produced on the 31^(st) day of the culture was observed between the two types of strains (TauT-transferred strain: 2050 mg/L; TauT+CSAD-transferred strain: 3383 mg/L). The amount of an antibody produced in the CSAD-transferred strain was 2170 mg/L. Moreover, since the survival ratio of a strain into which neither type of genes had been-transferred was low at the late stage of the culture, the maximum value of the amount of an antibody produced was 2006 mg/L on the 14^(th) day of the culture.

In order to examine the effect of a CSAD strongly expressing strain, four strains (into which 4 different genes had been introduced) were cultured in a 1-L jar feeding culture tank (2×10⁵ cells/mL in an initial stage). The culture medium was collected at appropriate times. (450×10⁵ cells were collected from each of the parent strain, TauT-transferred strain, and CSAD-transferred strain, and 200×10⁵ cells were collected from the TauT+CSAD-transferred strain. As a result, with regard to intracellular concentration, only the measurement value for the TauT+CSAD-transferred strain was corrected by multiplying by 2.25, and the thus corrected value was used.) The collected culture media were centrifuged to give culture supernatants used as samples for use in the subsequent measurement of amino acids in the culture medium. Thereafter, 1 ml of cooled sterile water having a protease inhibitor (Complete Mini; Roche Diagnostics; Protease inhibitor cocktail tablets) dissolved therein was added to the cell pellets, which were then pulsed on ice for 5 seconds using an ultrasonic cell disruptor (MISONIX ASTRASON MODEL XL2020). Such pulsing operation was then suspended for 5 seconds. This cycle was defined as one set of treatment, and a total of 12 sets of treatment were carried out, so as to completely disrupt the cells. The total amount of the solution after the treatment was applied to a centrifugal filtration unit to prepare filtrates with molecular weights of 5000 and less. The filtrates were used as samples for the measurement of intracellular amino acids. The absorbance at 570 nm of each sample was detected using a ninhydrin test solution-L 8500 set (Wako Pure Chemical Industries, Ltd.) and an improved model of a fully automatic amino acid analyzer (L-8500) manufactured by Hitachi, Ltd., and the obtained values were then compared with one another. Thus, the concentrations of various amino acids in the samples were determined. The concentrations of various amino acids and ammonia in the culture medium were directly measured. Thus, the determined concentrations were compared with one another on the μM order. With regard to intracellular concentration, since 1 mL of cooled sterile water was added to the cell pellets, followed by ultrasonic cell disruption, the measurement values of various amino acids and ammonia were converted to values per cell, and the thus converted values were compared with one another (FIGS. 11 and 13). With regard to the amino acid concentration ratio shown in FIG. 11, the detection value of intracellular β-alanine (in 450×10⁵ cells) derived from the CSAD strongly expressing strain at the initiation of the feeding culture in a 1-L jar was defined as 1, and this detection value was compared with the detection values for each strongly expressing strain at the initiation of the culture and on the 6^(th) and 12^(th) days after the initiation of the culture, so that each ratio was obtained. With regard to the ammonia concentration ratio shown in FIG. 13, the detection value of ammonia in the parent strain (in 450×10⁵ cells) at the initiation of the feeding culture in a 1-L jar was defined as 1, and this detection value was compared with the detection values at the initiation of the culture and on the 6^(th), 12^(th), and 18^(th) days after the initiation of the culture, so that each ratio was obtained. Moreover, changes over time in the concentrations of alanine and β-alanine in the culture medium during the culture were also measurement values derived from the aforementioned amino acid analysis.

As a result, no significant differences in intracellular amino acid concentration were observed by almost all amino acids among various types of gene-transferred strains. Only pHyg/CSAD, a CSAD strongly expressing strain, produced a high-yield of β-alanine that was not contained in the medium. This was also clear from the fact that when the concentrations of β-alanine and alanine in the culture medium were measured during the culture, the concentration of β-alanine significantly increased in the culture medium of the CSAD strongly expressing strain (FIG. 11-1). β-alanine is an organic osmolyte that acts to regulate osmotic pressure, and it has pH-buffering action and antioxidant action by itself. In addition, since such β-alanine is a precursor of carnosine (β-alanyl-L-histidine) which has a potent antioxidant activity, it can neutralize the fatigue substance lactate by its antioxidant action. Suppression of the amount of lactate produced as shown in FIG. 8 is considered to be brought about by β-alanine effect. Such β-alanine effect of the CSAD strongly expressing strain was significant in terms of survival ratio in feeding cultures in 1-L jar (FIG. 12). Moreover, intracellular ammonia in the TauT+CSAD-transferred strain was maintained at low concentration (FIG. 13), which is considered to contribute to high-yield production of antibodies.

The present invention can be applied to all types of antibody-producing cells.

Example 3 Survival Ratio-Maintaining Effect in the Presence of β-alanine

The parent strain and the CSAD-transferred strain were subjected to batch culture in a 50-ml shaker (2×10⁵ cells/mL in an initial stage) in the presence or absence of β-alanine (25 mM), and a comparison was made for their survival ratio. As FIG. 14 shows, on the 6^(th) day of the culture, the survival ratio of the CSAD-transferred strain capable of high-yield production of β-alanine was maintained in the absence of β-alanine (0 mM). In contrast, on the 6^(th) day of the culture, the survival ratio of the parent strain in capable of high-yield production of β-alanine decreased in the absence of β-alanine. The survival ratio of the parent strain on the 6^(th) day of the culture was maintained in the presence of β-alanine (25 mM), demonstrating the survival ratio maintaining effect of addition of β-alanine. However, from the survival ratio of the CSAD-transferred strain on the 7^(th) day of the culture, it was found that the survival ratio of the strain that became capable of high-yield production produced a large amount of β-alanine in the cells was maintained even in the absence of β-alanine (0 mM) at a level higher than that for the parent strain in the medium to which 25 mM β-alanine was added. Further, as shown in FIG. 15, even in the case where CSAD was co-transferred into a TauT-transferred strain to synthesize β-alanine in the cells, the survival ratio of the strain on the 6^(th) day of the culture was maintained in the absence of β-alanine (0 mM). On the other hand, as in the case of the parent strain of FIG. 14, a strain into which TauT was transferred but CSAD was not co-transferred maintained the survival ratio on the 6^(th) day of the culture in the presence of β-alanine (25 mM). There results demonstrate the survival ratio-maintaining effect of β-alanine that acts in cells. Thus, the results also demonstrate that the culture of cells capable of high-yield production of β-alanine is superior to the mere addition of β-alanine to a medium for cell culture in terms of the maintenance of the survival ratio of the cells or an increase in the yield of a desired polypeptide produced. Accordingly, the cell of the present invention which is capable of high-yield production of β-alanine is useful as a cell for producing all types of polypeptides such as an antibody or a physiologically active protein.

Reference Example 1 Cloning of CHO Cell-Derived Hamster Taurine Transporter Gene

Total RNA was extracted from anti-IL-6 receptor antibody-producing cells (A CHO DXB11 cell line into which an anti-IL-6 receptor antibody gene had been transferred) (Japanese Unexamined Patent Publication No. Hei 8-99902), and then cDNA was synthesized therefrom in a poly(A) dependent manner. Hamster taurine transporter (TauT) gene was obtained by PCR using as a template the cDNA fragmented with three restriction enzymes, SalI, XhoI and EcoRI. As PCR primers, those containing the 5′-end and the 3′-end sequence conserved between rat and mouse TauTs were designed. The nucleotide sequence of the cloned gene was determined. From its homology with other TauT genes of known species, the cloned gene was confirmed to encode hamster TauT (FIG. 16). The amino acid sequence of hamster TauT has high homology with mouse TauT (96% identity), rat TauT (96% identity) and human TauT (93% identity); it was predicted that hamster TauT is a transporter with 12 transmembrane regions (FIG. 17).

Reference Example 2 Increase in Viable Cell Density, Inhibition of Lactate Production and Increase in Antibody Yield, as Caused by Transfer of Hamster Taurine Transporter

CMV promoter expression plasmid pHyg/TauT (FIG. 18) was constructed by adding Kozak sequence to the hamster TauT (hereinafter, TauT) gene obtained by cloning in Reference Example 1. Control plasmid pHyg without pHyg/TauT or TauT gene was introduced by electroporation into the parent strain anti-glypican-3 antibody producing CHO cell (see WO 2006/006693). After selection of expression plasmid-transferred cells in the presence of hygromycin (400 μg/ml), all of the stably growing cell strains were expanded (pHyg/TauT: 8 strains; pHyg: 7 strains). TauT mRNA was prepared. Subsequently, 7 strains were confirmed to express TauT more strongly than the parent strain by the TaqMan method; they were selected as pHyg/TauT transferred cells. The mean mRNA expression level of these transferred cells (7 strains) was about 40 times larger than the control (7 strains). Cells of the total 14 strains were subjected to batch culture and fed-batch culture in 50 ml shaker flasks with an initial cell density of 2×10⁵ cells/ml. On day 7 of culture (late-stage), viable cell densities, lactate yields and anti-glypican-3 antibody yields in those strains were compared. In batch culture, growth inhibitory substances such as lactate accumulate in culture broth as cells grow and their growth is inhibited. However, the viable cell densities (FIG. 19) and lactate yields (FIG. 20) in pHyg/TauT transferred cells were superior to those in pHyg transferred cells (t test; p<0.05). With respect to anti-glypican-3 antibody yield, 4 out of the 7 strains of pHyg/TauT-transferred cell showed antibody yields higher than the highest yield in pHyg-transferred cell (FIG. 21). Further, since superiority of pHyg/TauT transferred cells in anti-glypican-3 antibody yield became more evident (t test; P<0.01; FIG. 22) in fed-batch culture, pHyg/TauT transferred T10 strain (which showed the highest growth ability among the above 4 strains) and the parent strain were subjected to fed-batch culture in 1 L jar. As a result, the viable ratio of T10 was maintained at 80% or more even on day 32 of culture (FIG. 23), with inhibited lactate production. Consequently, its anti-glypican-3 antibody yield achieved 2.9 g/L on day 35 of culture (FIG. 24). It was confirmed by flow cytometric analysis that TauT-transferred T10 cell was expressing TauT molecules on the cell membrane (FIG. 25). These results suggest that by artificially expressing hamster Taut, it is possible to raise the potential of antibody-producing cells and create strains capable of enhanced antibody production.

The present invention is applicable to any antibody-producing cell.

The fact that cells capable of producing antibodies in high yield were also obtained in a batch culture suggests that strong expression of TauT induced nonspecific integration of not only taurine but also β-alanine and many other amino acids. Organic osmolytes except taurine that have a cellular osmoregulatory function include β-alanine, alanine, glycine, proline, glutamic acid, glutamine, and aspartic acid, and the foregoing results predict that cells capable of high-yield antibody production can also be obtained by causing artificial expression of transporters for those organic osmolytes.

All publications, patent and patent applications cited herein are incorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

The present invention is applicable to production of polypeptides.

SEQUENCE LISTING FREE TEXT <SEQ ID NO: 1>

SEQ ID NO: 1 shows the nucleotide sequence of a gene encoding hamster cysteine sulfinic acid decarboxylase.

<SEQ ID NO: 2>

SEQ ID NO: 2 shows the amino acid sequence of hamster cysteine sulfinic acid decarboxylase.

<SEQ ID NO: 3>

SEQ ID NO: 3 shows the nucleotide sequence of a gene encoding rat cysteine sulfinic acid decarboxylase.

<SEQ ID NO: 4>

SEQ ID NO: 4 shows the amino acid sequence of rat cysteine sulfinic acid decarboxylase.

<SEQ ID NO: 5>

SEQ ID NO: 5 shows the nucleotide sequence of a gene encoding mouse cysteine sulfinic acid decarboxylase.

<SEQ ID NO: 6>

SEQ ID NO: 6 shows the amino acid sequence of mouse cysteine sulfinic acid decarboxylase.

<SEQ ID NO: 7>

SEQ ID NO: 7 shows the nucleotide sequence of a gene encoding human cysteine sulfinic acid decarboxylase.

<SEQ ID NO: 8>

SEQ ID NO: 8 shows the amino acid sequence of human cysteine sulfinic acid decarboxylase.

<SEQ ID NO: 9>

SEQ ID NO: 9 shows the nucleotide sequence of a gene encoding hamster cysteine dioxygenase.

<SEQ ID NO: 10>

SEQ ID NO: 10 shows the amino acid sequence of hamster cysteine dioxygenase.

<SEQ ID NO: 11>

SEQ ID NO: 11 shows the nucleotide sequence of a gene encoding rat cysteine dioxygenase.

<SEQ ID NO: 12>

SEQ ID NO: 12 shows the amino acid sequence of rat cysteine dioxygenase.

<SEQ ID NO: 13>

SEQ ID NO: 13 shows the nucleotide sequence of a gene encoding mouse cysteine dioxygenase.

<SEQ ID NO: 14>

SEQ ID NO: 14 shows the amino acid sequence of mouse cysteine dioxygenase.

<SEQ ID NO: 15>

SEQ ID NO: 15 shows the nucleotide sequence of a gene encoding human cysteine dioxygenase.

<SEQ ID NO: 16>

SEQ ID NO: 16 shows the amino acid sequence of human cysteine dioxygenase.

<SEQ ID NO: 17>

SEQ ID NO: 17 shows the nucleotide sequence of a gene encoding bovine cysteine dioxygenase.

<SEQ ID NO: 18>

SEQ ID NO: 18 shows the amino acid sequence of bovine cysteine dioxygenase.

<SEQ ID NO: 19>

SEQ ID NO: 19 shows the nucleotide sequence of a gene encoding hamster taurine transporter.

<SEQ ID NO: 20>

SEQ ID NO: 20 shows the amino acid sequence of hamster taurine transporter.

<SEQ ID NO: 21>

SEQ ID NO: 21 shows the nucleotide sequence of a gene encoding rat taurine transporter.

<SEQ ID NO: 22>

SEQ ID NO:22 shows the amino acid sequence of rat taurine transporter.

<SEQ ID NO: 23>

SEQ ID NO: 23 shows the nucleotide sequence of a gene encoding mouse taurine transporter.

<SEQ ID NO: 24>

SEQ ID NO: 24 shows the amino acid sequence of mouse taurine transporter.

<SEQ ID NO: 25>

SEQ ID NO: 25 shows the nucleotide sequence of a gene encoding human taurine transporter.

<SEQ ID NO: 26>

SEQ ID NO: 26 shows the amino acid sequence of human taurine transporter. 

1. A method for producing a polypeptide, in which cells capable of high-yield production of β-alanine and into which DNA encoding a desired polypeptide has been transferred are cultured to produce the desired polypeptide.
 2. The production method of claim 1, wherein the cells capable of high-yield production of β-alanine are cells into which DNA encoding a protein having enzyme activity for synthesizing β-alanine from aspartic acid has been transferred.
 3. The production method of claim 1, wherein the cells capable of high-yield production of β-alanine are cells into which DNA encoding cysteine sulfinic acid decarboxylase, glutamate decarboxylase, or a mutant thereof has been transferred.
 4. The production method of claim 1, wherein the cells capable of high-yield production of β-alanine are cells in which the ratio of the intracellular concentration of β-alanine on the 6^(th) or subsequent days after the initiation of the culture to that at the initiation of the culture is 2 or greater.
 5. The production method of claim 1, wherein the cells capable of high-yield production of β-alanine are capable of increasing the β-alanine concentration in the culture medium on 6^(th) or subsequent days after the initiation of the culture by 50 μM or more.
 6. A cultured cell, in which the ratio of the intracellular concentration of β-alanine on the 6^(th) or subsequent days after the initiation of the culture to that at the initiation of the culture is 2 or greater.
 7. A cultured cell, which is capable of increasing the β-alanine concentration in the culture medium on the 6^(th) or subsequent days after the initiation of the culture by 50 μM or more.
 8. A method for producing a polypeptide, in which cells that strongly express cysteine sulfinic acid decarboxylase and into which DNA encoding a desired polypeptide has been transferred are cultured to produce the desired polypeptide.
 9. The production method of claim 8, wherein the cells that strongly express cysteine sulfinic acid decarboxylase are cells into which DNA encoding cysteine sulfinic acid decarboxylase has been transferred.
 10. The production method of claim 8, wherein the cells that strongly express cysteine sulfinic acid decarboxylase further express a taurine transporter strongly.
 11. The production method of claim 10, wherein the cells that strongly express a taurine transporter are cells into which DNA encoding a taurine transporter has been transferred.
 12. The method of claim 9, wherein the cell is Chinese hamster ovary cells.
 13. The method of claim 8, wherein the desired polypeptide is an antibody.
 14. The method of claim 9, wherein the DNA encoding the cysteine sulfinic acid decarboxylase is any one of the following (a) to (e): (a) a DNA encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO: 2, 4, 6 or 8; (b) a DNA encoding a polypeptide which has an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 2, 4, 6 or 8 by substitution, deletion, addition and/or insertion of one or more amino acid residues and yet has cysteine sulfinic acid decarboxylase activity; (c) a DNA encoding a polypeptide having 70% or more amino acid sequence homology with the amino acid sequence as shown in SEQ ID NO: 2, 4, 6 or 8 and yet having cysteine sulfinic acid decarboxylase activity; (d) a DNA having the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5 or 7; (e) a DNA which hybridizes to a DNA complementary to a DNA having the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5 or 7 under stringent conditions and yet encodes a polypeptide having cysteine sulfinic acid decarboxylase activity.
 15. A DNA encoding cysteine sulfinic acid decarboxylase, which is any one of the following (a) to (e), provided that DNA having the nucleotide sequence as shown in SEQ ID NO: 3, DNA having the nucleotide sequence as shown in SEQ ID NO: 5, and DNA having the nucleotide sequence as shown in SEQ ID NO: 7 are excluded: (a) a DNA encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO: 2; (b) a DNA encoding a polypeptide which has an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 2 by substitution, deletion, addition and/or insertion of one or more amino acid residues and yet has cysteine sulfinic acid decarboxylase activity; (c) a DNA encoding a polypeptide having 97% or more amino acid sequence homology with the amino acid sequence as shown in SEQ ID NO: 2 and yet having cysteine sulfinic acid decarboxylase activity; (d) a DNA having the nucleotide sequence as shown in SEQ ID NO: 1; (e) a DNA which hybridizes to a DNA complementary to a DNA having the nucleotide sequence as shown in SEQ ID NO: 1 under stringent conditions and yet encodes a polypeptide having cysteine sulfinic acid decarboxylase activity.
 16. A polypeptide which is any one of the following (A) to (E), provided that a polypeptide having the amino acid sequence as shown in SEQ ID NO: 4, a polypeptide having the amino acid sequence as shown in SEQ ID NO: 6, and a polypeptide having the amino acid sequence as shown in SEQ ID NO: 8 are excluded: (A) a polypeptide having the amino acid sequence as shown in SEQ ID NO: 2; (B) a polypeptide which has an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 2 by substitution, deletion, addition and/or insertion of one or more amino acid residues and yet has cysteine sulfinic acid decarboxylase activity; (C) a polypeptide having 97% or more amino acid sequence homology with the amino acid sequence as shown in SEQ ID NO: 2 and yet having cysteine sulfinic acid decarboxylase activity; (D) a polypeptide encoded by a DNA having the nucleotide sequence as shown in SEQ ID NO: 1; (E) a polypeptide encoded by a DNA which hybridizes to a DNA complementary to a DNA having the nucleotide sequence as shown in SEQ ID NO: 1 under stringent conditions and yet encodes a polypeptide having cysteine sulfinic acid decarboxylase activity.
 17. A recombinant vector comprising the DNA of claim
 15. 18. A cell into which the DNA of claim 15 has been transferred.
 19. A cell which has a transferred DNA encoding cysteine sulfinic acid decarboxylase and a transferred DNA encoding a desired polypeptide.
 20. The cell of claim 19, which further has a transferred DNA encoding a taurine transporter.
 21. A cell which has a transferred DNA encoding cysteine sulfinic acid decarboxylase and a transferred DNA encoding a taurine transporter.
 22. A cell capable of high-yield production of β-alanine which can be obtained by transferring a DNA encoding cysteine sulfinic acid decarboxylase.
 23. The cell of claim 22, which further has a transferred DNA encoding a desired polypeptide. 